Despite growing understanding of the regulation of skeletal muscle differentiation, the molecular basis for a fundamental component of this process - concurrent, irreversible cell cycle arrest - remains largely unknown. Although the MyoD family of myogenic regulators can initiate the skeletal muscle differentiation program, it is unclear whether these proteins also induce cells to terminally withdraw from the cell cycle. Because muscle gene expression is itself linked to a G0 arrest in differentiated myotubes, it would seem reasonable to propose that the factors which promote cell proliferation may negatively regulate the functional activity of the MyoD family of proteins. Hence, the basic hypothesis for the present proposal is that cell cycle regulatory factors may also regulate the activity of a key myogenic regulatory factor, MyoD. AS such, the primary event which initiates terminal differentiation in skeletal myoblasts may be one which causes cells to arrest in the G0 phase of the cell cycle. Because modulation of the activity of cyclin- dependent kinases (cdks) has been implicated in controlling cell cycle progression, a series of experiments have been initiated to investigate the effect of cyclin-dependent kinase activation on MyoD function. The specific objectives of these experiments are to determine (1) which regulators of cell cycle progression (cyclins, cdks, p21, p16INK4) are present in proliferating myoblasts versus terminally differentiated myotubules arrested in the G0 phase of the cell cycle, (2) whether ectopic expression of specific G1 cyclins can inhibit full muscle differentiation in vitro. (3) what is the mechanism of a G1 cyclin- mediated inhibition of MyoD function, and (4) whether cyclin-dependent kinase activity can be manipulated in vitro to allow terminal differentiation and cell cycle arrest under conditions where MyoD is present but functionally inactive (i.e. in proliferating myoblasts, oncogenically transformed myoblasts, and rhabdomyosarcoma cells). The ultimate goal of these studies will be their effective extension to clinical settings where one would like to induce terminal muscle differentiation and cell cycle arrest (e.g. rhabdomyosarcoma) and to clinical settings where one would like to allow terminally differentiated skeletal myocytes to re-enter the cell cycle (e.g. muscle atrophy and injury).